Commercially available DNA sequencing kits, such as the ABI PRISM® BigDye® Terminator v.1.0, 1.1, 2.0, 3.0 and 3.1 Ready Reaction Cycle Sequencing Kits available from Applied Biosystems, Inc. utilize fluorescently-labeled molecules or dye terminators as a component. For example, the dye terminators, deoxynucleoside triphosphates, sequencing enzyme, magnesium chloride and buffer are premixed and are suitable for performing fluorescence-based cycle sequencing reactions on single-stranded or double-stranded DNA templates and on polymerase chain reaction fragments.
Because dye terminators are not natural substrates of DNA polymerase, high concentrations generally must be provided relative to the natural dNTP substrates to ensure their incorporation into the polymerizing sequencing products. The unincorporated fluorescently-labeled molecules, however, are difficult to remove if present in high concentration. If they are not adequately removed, the unincorporated fluorescently-labeled molecules can interfere with downstream analysis (e.g., DNA sequencing), such as by co-migrating with short sequencing products during electrophoresis. Indeed, these molecules have a tendency to form insoluble complexes at high concentrations. They are particularly problematic in reactions that utilize high concentrations of sequencing chemistries (e.g., 1×, ½× and ¼× strength reactions).
Conventional methods for removing unincorporated dye terminators from sequencing reactions prior to electrophoresis involve alcohol precipitation and gel filtration. However, salts compete with sequencing products for electrokinetic injection onto capillary sequencing instruments and also must be removed. Ethanol precipitation has poor salt removal capabilities which detracts from its utility as a method for preparing samples prior to capillary electrophoresis because the efficiency of electrokinetic injection of sequencing products is inversely proportional to the salt concentration. Gel filtration is a centrifuge-based method which is difficult to automate, which is important for high throughput DNA sequencing.
Another method of removing unincorporated fluorescently-labeled molecules such as dye terminators involves using MultiScreen® or Montage™ 384-SEQ ultrafiltration plates commercially available from Millipore Corporation. These plates are fully automatable and provide a cost-and-time efficient alternative to conventional ethanol precipitation for dye-terminator removal. They operate by vacuum filtration, thereby eliminating the need for centrifugation, ethanol drying steps and manifold disassembly routines. Solvents such as formamide or EDTA aid in the solubilization of dye terminators and prevention of aggregate formation. The sequencing products are purified by filtering to dryness and then washing salts an dye terminators to waste. The purified sequencing products are then resuspended from the membrane surface and are ready for introduction into a DNA sequencer.
However, the introduction of new sequencing chemistries by various manufacturers continues to present purification challenges. In addition, although current ultrafiltration technology provides substantially purified DNA sequencing products at reaction strengths of one microliter of sequencing reagent per DNA sequencing reaction (⅛th BDT v3.0), at higher concentrations artifacts known as dye blobs become apparent on electropherograms. These artifacts are also commonly visible using other clean-up methods such as gel filtration and alcohol precipitation despite specific modifications to the protocol recommended by the manufacturer to eliminate them.
It therefore would be desirable to provide a cost-effective and efficient solution for reducing or eliminating unincorporated fluorescently-labeled molecules and residual salts from sequencing reactions.